Fluorescent double stranded DNA binding dyes such as ethidium bromide have been used for a long time for staining nucleic acids within gel matrices that have been subjected to electrophoresis.
Equally important, fluorescent double stranded DNA binding dyes as have been used in the art for real time monitoring of nucleic acid amplification such as PCR or melting curve analysis. The respective amplification product is detected by a fluorescent DNA binding dye which emits a corresponding fluorescence signal upon interaction with the double-stranded nucleic acid after excitation with light of a suitable wavelength. The at least partially intercalating dye SYBRGreenI (Molecular Probes, Inc.) has been proven to be suitable for this application.
Due to the fact that real time amplicon detection with this format can not discriminate between specific products and amplification artefacts such as primer/dimers, a subsequent melting curve analysis is usually performed. After completion of the PCR reaction, the temperature of the sample is constitutively increased, and fluorescence is detected as long as SYBRGreen is bound to the double stranded DNA present in the samples. Upon dissociation of the double stranded DNA the signal decreases immediately. This decrease is monitored with an appropriate fluorescence versus temperature-time plot such that a first derivative value can be determined, at which the maximum of fluorescence decrease is observed. Since primer/dimer double stranded DNAs are usually short, dissociation into single stranded DNA occurs at lower temperatures as compared to the dissociation of the double stranded specific amplification product.
There are several fluorescent double stranded DNA binding dyes known in the art which can be used as a detecting agent for real time PCR and melting curve analysis. The most prominent and most frequently used example is SYBRGreen I which is an asymmetric monomeric cyanine dye comprising an N-alkylated benzothiazolium ring system which is linked via a monomethine bridge to a pyridinium or quinolinium ring system (U.S. Pat. No. 5,658,751). More precisely, SYBRGreen has the chemical formula [2-[N-(-3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]+ (Zipper, H., et al., Nucl. Acids Res. 32 (2004) e103). In the related dye PICOGREEN (Molecular Probes, Inc.), the chemical formula reads [2-[N-bis-(-3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]+.
U.S. Pat. No. 4,937,198 discloses a related fluorescent DNA binding dye having the chemical formula 3-methyl-2-[(3,7-dimethyl-6-purinylidene)-methyl]-benzothiazolium, which has been used successfully for nucleic acid staining in cell-based assays. Further dyes and respective analytical means are disclosed in Moreda, W. and Forrester, A. R., Tetrahedron 53 (1997) 12595-12604.
WO 04/38038 discloses examples of fluorescent dsDNA binding dyes which are based on a pyrimidinium ring system and a benzothiazolium which are connected by a methine bridge. In contrast to SYBRGreen, it is possible to use these dyes for detecting PCR amplification products under saturation conditions, i.e. at high concentrations where each DNA molecule already binds a maximum number of fluorescent molecules. Within US 2005/233335, the same group of inventors discloses further specific examples of fluorescent molecules comprising a pyrimidine moiety and a benzthiazolium moiety.
Many of the disclosed dyes have successfully been used for real time PCR and amplicon melting curve analysis. However, all fluorescent double stranded DNA binding dyes described in the art either have a limited stability and/or are of only limited use for improved methods of melting curve analysis. For such improved methods of melting curve analysis which may be applied for mutation scanning (WO 04/38038), the effects of minor temperature changes with respect to the conformational status of the target nucleic acid are being analyzed.
Thus it was an object of the present invention to provide an improved double stranded DNA binding fluorescent dye with increased stability and improved performance during DNA melting curve analysis.